e frontiers in Pharmacology OPEN ACCESS Ecltod by: Govern Vt, reat of Ctra, tay ‘Reviewed by: Pata Kestamacy, Unversity of Kansas, USA ec Vik, eras Unveraty Prague, ‘Correspondence: ‘na aD ctnesn papueuoporacs "Present aderess: Dish Babu, Kate Group Gants fe Prsmacy ‘a Healy Rosorch Foca of pramacy and Pramacoutcat ‘Soences, Unversity of Abra ‘anonton, AB, Canada Speciaty section: xpenmantalPramaccicgy an One Dessovey, a secton oft ura FrontersnPramacatogy Rocetved 12 November 2016 ‘Accepted: (6. 2017 Puntsned 08 Fay 2017 citation abu 0, Lack @ Motil Rand Lele FA 2017) Oferta Etec (of CORY.2 anc CORI-01 Meno resin Epthatal HODEK Cals Und Ovcatne Sts, Front Pharmacl 8371, be 10. 880har 201700031 ORIGINAL RESEARCH. sublet 08 Faby 2017 8 Differential Effects of CORM-2 and CORM-401 in Murine Intestinal Epithelial MODE-K Cells under Oxidative Stress Dinesh Babu’, Georges Leclercq?, Roberto Motterlini? and Romain A. Lefebvre" Hoymans tut of Phamacoloy Faculyof Maia and Heath Sconces, Ghant Universi), Gran, Bolum, Dazrinent of Cea Chany. crabby and inmunotoay, Faculty of Madcne and Heh Sowrees, Ger Unversity Gon, Bote * INSERM UD55,Facly of Moco, Equipo 2 and Unesty Pars Est, Crit Franco Carbon monoxide (CO)-releasing molecules (CO-RMs) are intensively studied to provide cytoprotective and ant-infammatory effects of CO in inflammatory conditions including intestinal infammation. The water-soluble CORM-A1 reduced apoptosis and NADPH oxidase (NOX)-derived reactive oxygen species (ROS) induced by tumor neorosis factor (TINF)-a/cycloheximide (CHX) in mouse MODE-K intestinal epithelial ces ECs}, without influencing TNF-a/CHX-induced mitochondrial superoxide anion (03~). The aim of the present study in the same model was to comparatively investigate the influence of lipid-soluble CORM-2 and water-soluble CORM-401, shown in vitro to release more CO under oxidative conditions. CORM-2 abolished TNF-a/CHX induced total cellular ROS whereas CORM-401 partially reduced it, both partialy reducing TNF-a/CHX- induced cell death. Only CORM-2 increased mitochondrial O3~ production after 2 h of incubation. CORM-2 reduced TNF-a/CHX-, rotenone- and antimycin-A induced mitochondrial O$~ production; CORM-401 only reduced the effect of antimycin-A, Co-treatment with CORM-401 during 1 h exposure to HO2 reduced H202 (7.5 mM- induced ROS production and cell death, whereas CORM-2 did not. The study llustrates the importance of the chemical characteristios of diferent CO-RMs. The lipid solubility of CORM-2 might contribute to its interference with TNF-a/CHX-induoed mitochondrial ROS signaling, at least in mouse IECs. CORM-401 is more effective than other CO-RMs Under H,02 induced oxidative stress conditions. Keywords: carbon menoxide-oleasing molecules, hydrogen peroxide, intestinal epithe ‘oxidative stress, reactive oxygen species, sob, TNF-o/CHX ‘mitochondria, INTRODUCTION Acute and chronic gastrointestinal (GI) inflammatory disease conditions are associated with persistent oxidative stress originating from increased reactive oxygen species (ROS) that are known to initiate and perpetuate inflammation (Bhattacharyya et al, 2014 Mittal et al, 2014). Oxidative stress-induced epithelial cell damage and increased intestinal permeability toward luminal commensal bacterial material, which activates mucosal immune cells and triggers muscular inflammation, can contribute to the severity of acute GI disorders as observed in animal models of septic ileus, necrotizing enterocolitis and ischemia/reperfusion injury (Anup et al., 1999 de Winter et al, 2005; Baregamian et al., 2009; De Backer et al, 200%; Guan et al., 2008). Excessive production of ROS and mucosal injury has also been reported in animal models of inflammatory bowel disease (IBD) (Ahn et al, 2001; Reifen et al, 2004; Cetinkaya et al, 2005) and in colonic tissue of patients with ulcerative colitis (Oshitani etal, 1993; Nishikawa etal, 2005), Fronts in Phamacciogy | wn ontersinog 1 Fetrury 2017 | our 8 | arteleabu et ‘Tumor necrosis factor (TNF)-a.is one ofthe early inflammatory ‘mediators thought to play an important role in epithelial barrier dysfunction by inducing intestinal epithelial cll (IEC) apoptosis ROS play an important role in TNF-a-induced apoptotic cell death of IECS (Jin et al, 2008; Baregamian et al., 2009; Babu tal, 2012). The nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) family and the mitochondrial electron transport chain (ETC) ate the two major ROS: producing sources involved in TNF-a/cycloheximide (CHX)-induced cell death in rat IEC-6 cells (Jin ct al, 2008) and in mouse MODE-K cell Babu et al, 2015b). In MODE-K cells in particular, complexes [and Il of the mitochonrial ETC were found to be the main sites of superoxide anion (O$~) production in addition to NOX GBabu et al, 2015b). As the endogenous antioxidant defense system does not seem sutficient to counteract TNFc-induced ROS production, neutralizing excessive ROS production might be an elective therapeutic strategy to reduce intestinal barrier dysfunction during Gl inflammation. “The stress-responsive protein heme oxygenase (HO)-1 i up regulated by oxidative stress and inflammatory signals, and it generates biliverdin, a powerful antioxidant, and carbon ‘monoxide (CO), which exerts antioxidant, ant-inflammatory and cytoprotective effects (Ryter et al, 2006; Motterlini and Otterbein, 2010), Inhalation of CO gas has been successfully applied in animal models of inflammation and oxidative stress but translation to humans might be difficult (i et als 2016). From pharmacological and therapeutic perspectives, small molecules capable of delivering controlled amounts of CO to biological systems have therefore been developed to. mimic the intrinsic beneficial effects of CO (Moiterlini et al, 2002; Sawle et al, 2005; Mottelini and Otterbein, 2010). These compounds, known as CO-releasing molecules (CO-RMS), have been extensively studied and belong to two major classes (1) metal carbonyl complexes containing ruthenium, manganese, of molybdenum, which carry CO bound to the transition metal and (2) boranocarbonates, which do not contain transition rmetals but the metalloid boron and release CO spontaneously in physiological conditions, the rate of release being affected by changes in pH. While the original lipophilic CO-RMs such a8 CORN ([Mnx(CO))) and CORM-2 ({Ru(CO);Cl]2) have to be dissolved in organic solvents such as dimethyl sulfoxide (DMSO) (Motterlini etal, 2002), water-soluble CO RMs such as CORM-3 ({Ru(CO)sCl(glycinate)]) and CORM. Al (Naz[HsBCOp)) were subsequently developed (Clark et al. 2003: Motterlini et al, 200Sb). These compounds have been shown to be pharmacologically active in limiting cellular and tissue dysfunctions in a number of pathological disorders associated with inflammation and tissue injury (Motterlini and Forest, 2014). The inhibitory effect of CO and CO-RMs on cytokine-induced changes inthe intestinal epithelium might also contribute to their beneficial effect in acute GI inflammation such as postoperative ileus (Babu et al, 2015e) and in chronic GF inflammation such as IBD (Takagi et al, 2015). Although the exact mechanism(s) for the antioxidant and cytoprotective effect of CO is (are) still under investigation, emerging evidence indicates that the beneficial properties of CO may be linked to its ability to bind to hemoproteins, such as NOX and (CO-RMe under Oxane Ses Conctons ‘mitochondrial complexes in different tissues (Taille et al. 2005; Bilban et al, 2008). At the mitochondrial level, CO was shown to induce a transient burst of mitochondrial ROS (O$-) that is thought to promote a preconditioning state, enabling it to counteract subsequent oxidative stress (Taille et al., 2005; Chin et al, 20075 Vieira et al, 2008). We previously showed that the water-soluble CORM-AI reduced both TNF-a/CHX- induced ROS production and apoptosis in MODE-K IECs (Babu et al, 2012, 2015a). At cytoprotective concentrations, CORM-AI per se did not induce mitochondrial OF ; however, CORM-AL inhibited NOX-derived ROS production, but not mitochondrial O}- production, after challenging MODE-K IECs with TNE-a (Babu et al, 2015a). This absence of an effect at the mitochondrial level might be related to the ‘water-soluble properties of this CO-RM, which provent its penetration (0 sites of ROS production in mitochondria. By contrast, the lipophilic CORM-2 was shown to induce ROS production from mitochondria in human bronchial smooth :uscle cells (Taille et al, 2005). The cytoprotective properties ‘of CORM.2 in IECs and its effect on cellular targets mediating ROS production have yet to be investigated, CORM-401 [Mn(CO),{S,CNMe(CH;CO;H)}| is recently developed water soluble CO-RM that releases up to three equivalents of CO per mole of compound, in contrast to CORM-Al which releases one equivalent of CO (Motterlini et al, 2005a; Crook et al,, 2011; Fayad-Kobeissi et al, 2016). Moreover, the rate fof CO release from CORM-401 in cell-free int vitro systems is accelerated in the presence of biologically relevant oxidants, such as hydrogen peroxide (H2O2) (Fayad-Kobeissi et al, 2016), In view of the above considerations, in the present study, we compared the cytoprotective effects of CORM-2 and CORM-401 in MODE-K IECs under oxidative stress conditions, evaluating their effects on oxidant-generating system(s). For the protocol with a high concentration of Hy03, also CORM-AL was ‘compared as this was not investigated in our previous study with the compound (Babu et a., 2015a). The major characteristics of, the three CO-RMs are summarized in Table 1 MATERIALS AND METHODS: Chemicals and Reagents Reagents for cell culture, including Dulbecco's modified Eagl’s medium (DMEM), fetal bovine serum, pencilinfstreptomycin and GlitaMAX” were obtained from Gibco BRL. (Grand. land, NY, USA). Carboxylated analog of 27 dichlorodinydroflorescein diacetate acetyl ester (catboxy H:DCEDA), MitoTracker Deep Red FM, MitoTracker Green FM, MitoSOX Red, Sytox Green, Sytox Red. and tetramethylrhodamine methylester (IMRM) were purchased from Molecular Probes - Invitrogen (Carbad, CA, USA). Recombinant murine TNF-a was purchased from R&D systems (Minneopolis, MN, USA). Antimycin-A, CHX, CORM-2, DMSO, HO> and rotenone were purchased from Sigma (St Louis, MO, USA). CORM-Al and CORM-401 were synthesized as previously described (otter etal, 20056; Crook etl, Fetrury 2017 | our 8 | arteleabu et TABLE 1 | Structure and characteristics of CO-RMS stucid. (CORN under Oxia Ses Conctons Compound (Chemica structure SSoubitty In vivo nats (i) at ‘Maximal amount of CO t37'C, pl =T4 released per mole cone uso tye mn Mote ot one FuCOCe 2KD} ye < 1m Denar eal, 2012 ccont.so1 20 ye increased ROS production by 430-505% compared with control (Figures 2A,C) indeed without inducing cell death (Figures 2B,D). Both CORM, 2 and CORM-401 significantly attenuated the Hz02-induced increase in otal ROS production (Figures 2A,C). [As the release of CO from CORM-401 has been shown, to be accelerated by increasing concentrations of oxidants, a concentration-response curve to HzO2 higher than I mM was constructed, assessing both intracellular total ROS production and cell death (Figures 2E,P). From these experiments, incubation of eels with 7.5 mM HO, for 1 h was selected as this condition induced a evel af cell death similar to that induced by "TNF-a/CHX for 6b i. 57 + 4% (Figure 2F) this concentration ‘of HO» increased ROS production by 2900% relative to the control (Figure 2), ‘When the standard treatment protocol was used for CORM. 2 (ie, pre-treatment for 1h before exposure to H2O2 and ‘then co-treatment during exposure to H2O2), HOz-induced ROS production and cell death were significantly reduced by 34 10% and 41 + 4%, respectively (1 = 3; Figures 3A,B). By contrast, either pre-treatment with CORM-2 for 1 h or co- treatment with CORM-2 during the I h exposure to HaOa did not affect Hz0,-induced ROS production (Figure 3A) or cell death (Figure 3B), Pre-treatment of cells with CORM-401 followed by co-treatment with CORM401 and HO, was even more elfective as it decreased both H,O2-induced ROS produ and cell death by 75 + 5% and 72 +E 6%, respectively (nr Figures 3C,D). Interestingly, and in contrast to the observations ‘made with CORM.2, just pre-treating cells with CORM-401 for 1 h or just co-treating them with CORM-401 during the Th exposure to HO» was sufficient to significantly reduce H03-induced ROS production and cell death, although to a lesser extent compared with the pre- plus co-treatment protocol (r= 3; Figures 3C,D). Another water-soluble CO-RM, CORM, AA1 showed similar effects as CORM-2 (11 = 3; Figures 3E,B). Effects of CORM-2 and CORM-401 on TNF-a/CHX-Induced Changes in Mitochondrial O$~ Production and Cell Death The influence of CORM-2 and CORM-401 on TNE-aiCHX. induced mitochonctrial O$~ production was assessed by using Me under Oxtne Stress Coens the fluorescent probe MitoSOX Red in conjunction with Sytox Red, to discriminate viable ws. dead cells, ina single experimental setup. Exposure of cells to TNB-a/CHX increased mitochondrial O}> production by approximately 200% relative to the control, and treatment with CORM-2 reduced this effect (Figure 4A) alongside a concomitant decrease in TNF-a/CHX-induced cell death (Figure 4B). iCORM-2 had no effect per se nor did it influence the TNF-a/CHX-induced changes (data not shown). By contrast, although CORM-401 significantly reduced ‘TNF a/CHX-induced cell death (Figure 4D), it did not influence TNE a/CHX-induced mitochondrial O$~ production (Figure 4C). Incubated for 7 h, neither CORM-2 nor CORM-401 per se inereased mitochondrial O}~ production (Figures 4A,C). When ‘eating the cells for 1, 2, or 3h, CORM-2 promoted a significant inerease in mitochondrial Of when incubated for 2 h, whereas CORM-401 did not change mitochondrial Of~ production (Figure 48); also iCORM-2 was without effect (data not shown). Treatment with CORM-2 or CORM-401 did not result in an increase in intracellular total ROS production at these time points (Figure 4). Effects of CORM-2 and CORM-401 on Mitochondrial Complex I- and Complex Ill-Induced Changes in Mitochondrial O$" and Cell Death Rofenone (7.5 uM), an inhibitor of mitochondrial complex 1, induced an increase in mitochondrial Of" level comparable to tat induced by TNF-a/CHX (Figures 5A,C), without affecting cell survival (Figures SB,D). The concentration of 73 uM rotenone was selected based on a concentration-response study of th effet of rotenone on mitochondrial Of” , when incubated for 6 hto mimic the exposure time to TNF-a/CHX (Babu et al, 20158). Treatment with CORM-2 nearly abolished rotenone induced mitochondrial Of” production, whereas CORM-401 had no elec (Figures 54,0) The influence of CORM.2 and CORM-401 on mitochondrial Of production atthe level of mitochondrial complex TIT was investigated by use of the complex Ill inhibitor antimycin-A an agent well knovn to induce OF” production at this site ‘Antimycin-A was used at 10 4M for 6 b (Babu et al, 2015a) as this concentration did not induce cell death in MODE: K calls (see Figures SRH), but increased mitochondrial Of production toa level comparable to that induced by TNF-a/CHD (see Figures 5E,G), Hoth CORM-2 and CORM.-401 significantly decreased (Figures 5E,G) antimycin-A-induced mitochondrial OF production. 1CORM-2 had no influence on the rotenone- oF antimyein-A induced inctease in mitochondrial OF levels (data not shown), Effects of CORM-2 and CORM-401 on TNF-c/CHX-Induced Changes Mitochondrial Dysfunction and Mitochondrial Membrane Potential (Wm) of MODE-K Cells Double staining of MODE-K cells with two different mitachondria-specific dyes to distinguish actively respiringi ns too + z _ 5 600: ge6 se [| Bs i ts & 200- a 2 om. : 2° & fs R 7 od : D ~ eo ns : - Bee ce 32° J #8 400. * Bea Bs £ : 200. & ey # e F 5000 + : = 4000 EE 2000 5! ano : = 1000- ie] : = = = a of pee ae +H202 (1h)a e 4000 > z zEe $500 g 23 § 8 2000. BS 00 E 1000: £2 20 0. 8 4h coRma. th conma gn foORM2 gn feoRte2 lice [iyo c > 4000 80 F000 60 ge & 8 2000 40 as 1000- 20 ©. °. ‘4h coRMa01 ‘th cORMAD1 gn fooRMsot gn fooRMsot lio, [nos e F 4000 pm tee ue 25 3 i 2 2000 22 «0 as xs & 1000. $2 2 e o- eG 1h coRmat th coma — + — + + = ssfcommet te co ee ee es wor asested with carbo’ ¢OCFDA DCF), and gated cn the atl, Syox Reeve ponuain. nacalkar fl ROS lve re DCF MF ol cosh expected as Sok dane, Cntr cal were neon wth onan re mam aloe: he eet of ORI, CORN oF CORW-AT per se ls ested. Moen & SEM of then copendentexpararte. “P< 0.05 ve, canta. "P< 005 = H:0: ano. FIGURE 9 es of CORM-2 (AB), CORM0 (6.0) or CORW-AT (EF) (MitoTracker Deep Red FM-positive) and total (MitoTracker compared with untreated control cells (35-40% vs. 4-6%; Green FM-positive) mitochondria revealed an increase in Figures B,D), Treatment with CORM-2 (Figures 6A,B) the number of respiration-interrupted mitochondria in the significantly attenuated the TNF-a/CHX-induced increase in P2-gated region upon treatment of cells with TNF-a/CHX — dysfunctional mitochondria, whereas treatment with CORM-401 (representative examples in the third panel of Figures 6A,C) did not have any effect on this parameter (Figures 6C,D).400. i 2 = s00. : * i 8 200 5 22 to 5 l : < ey = CA A Ae oe ” : é oo ._. : x 8 200- gs # §5 3m gE 100 j e - oe ® s eS co SOF fs 8 s s : ' 20 : Em . 22 = 5 BS wo 52 as a £® wo 2 o o- IPF PoP PS SS oof Pas ESE SSP ESS SSPE ESS tna Sane aan Gu ean Guan FRGURE «| A-0) fc of OM IA CORO" (0) on TN Cn mbna OF ptcon andal h PMODE cs MODE Inca ots ROS (measur os Geena Figure 1. Mean& < oe et (feaues yo.) aN pee xOSeHNabu et Me under Oxctne Sees Coens Mitotracker Deep Red > wigs gy ) aa ‘wells with dysfunctos ‘mitochondria ‘CORMAOI + TNF a/CHX Mitotracker Deep Red ‘Mitotracker Green FM " : 2 g 40- = He a i : OT CPL E ae FIGURE 6 Etocs of CORM-2 (AB) or CORN-40! (6,0) an TNF w/CHK-rdusad mitochon dyeuncten in MODE: cls, MODE: cols wer tele for vith TNF (1 nmr (10 wate absence ad presence ef CORN (0 Mot CORW-01 50 RM, added 1h bere TNF-alCHXTherumber of eas wn respng mcenonara was assessed wh MS Tacks Green FM (FL FTC) ae Ma Wacker Deap ed FM (FLA; APG) Cena cas were cubated wh ‘une mom are: De eet of CORM2 cr CORN-01 perso as. a0 Tested. (A.C) Frenne dl oso the ow cemeto anase aowhg he ‘tect of CORM.2 (A or COFB-101 () er BoTracker Croan FM ets mcehondtal ve MoTiacker Daop Red FM (athe espeng mtcchordi ucescehce ot able cats ater ueatmant wn TN-a/CHK Cs te gated ragen(P2) conan respuaion-rtanupted mocnenaa (BB) Quarticaton of Tow cvomere asronents oxrensed a of cal wih shanna ated) or exper wh CORM-? () aed COPM-401 (O) Mean + SEM ote independent lmpermers. "P< 0.05 econ, "P< 005 ve. TNF-a/CHX sone,* 5 § oo . 60: * £2 ot * ge gif # ea” * ee # Fea 28m #5 A £ & z 0- Saas - - 2 rs . 3 & ss . 3 . 7 22 uo * gs = & 3S 3 | om ee = Ag Es FIGURE 7 | Etecs of CORM-2 {AB} or CORM-A01 (¢,0) on TNF-w/G+ died changes mitochon membrane potenti and al deat MODE: cal MODE: cole wer ted or Gh th TNF-a ngmw/CH (10 gi, outed er nthe aboenon orl presen of CORM2 40 pM) or CORN (GO nM, acces 1 hbo INFalCHK Moca merrrane ptetal was assessed wth TMAM, and gate onto val, Syox Groen-negaive poration, Mtocroncna memtrane pateralsexoresead ea of cas wth Gepotrzed maocronca ae measured by THEM ing, cl ath is express a % Syox ‘Geen potty. Contra ols wera nabs wth serum ea mecum sere ti ecto COR 2 ar CORNE-AO paso ae ots. Moan = SEM eras 0.05 conta, "P< 0.08 v, TNFwICH aloe, We next studied the influence of CORM-2 and CORM. 401 on TNFa/CHX-induced changes in Va using a potentiometric dye, TMRM, TMRM facilitates analysis of cell death simultaneously in combination with the cell death marker Sytex Green. Approximately 7-8% of control cells, showed depolarized mitochondria, Treatment of cells with TNF-a/CHX caused approximately 60% depolarization of mitochondria (Figures 7A,C). CORM.2 decreased the number ‘of depolarized cells by TNF-a/CHX to 39% (Figure 7A) but CORM-401 did not influence this parameter (Figure 7C), ‘whereas their inhibitory effect on TNF-a/CHX-induced cell death was confirmed (Figures 7B,D). ICORM-2 had no influence on the TNF-a/ changes in both assays (data not shown). CHX-induced DISCUSSION TNF-a/CHX-induced apoptosis of MODE-K cells is associated with a marked production of ROS, the major sources beingabu et NOX and mitochondrial ETC complexes { and If (Babu et al, 2012, 2015b). One of the possible cellular mechanisms by ‘which CO confers cytoprotection is via modulation of ROS production (Peers etal, 2015). We previously found that water- soluble CORM-AI reduces TNF-a/CHX-induced apoptosis by inhibiting NOX-derived ROS production but not mitochondeial Of production induced by TNF-a (Babu et al, 2012, 2015a)s treatment of MODE-K cells with CORM-A1 at concentrations that display a cytoprotective effect did not change basal mitochondrial Of production, even in the first hours after its administration. These results suggest that CORM-A1 is unable to interfere with sites of ROS production at the mitochondrial evel this result might be attributed to the water-soluble property of this CO-RM impairing its penetration into the cell and/or through the inner mitochondrial membrane (Horton et al, 2008). In the present study, we investigated the influence of the lipid-soluble CORM-2 under oxidative stress conditions in comparison with the newly developed water-soluble CORM- 401, which was shown to release more equivalents of CO under ‘oxidant conditions (Fayad-Kobeissi etal, 2016); see Table 2A for qualitative summary of the effects of CORM-2 and CORM- 401 in basal conditions and vs. TNE-a/CHX, rotenone, and JA treatment. anti Mechanism of Action of CORM-2 during TNF-o/CHX-Induced Oxidative Stress In contrast to previous results with CORM-A1 (Babu et al 2015a), pre- and co-incubation of MODE-K cells with CORM-2 abolished TNF-a/CHX-induced total cellular ROS production and markedly diminished cell death. CORM-2 also reduced TNF-a/CHX- induced mitochondrial O$~ production suggesting that CO released from CORM-2 can interfere at mitochondrial ROS production sites. CORM-2 indeed also reduced rotenone and antimycin-A-induced mitochondrial O$~ production demonstrating its capacity to mitigate complex I and complex Ul-derived ROS. The observed effects of CORM-2 can be attributed to the effect of CO released from it, as \CORM-2 both perse and in the presence of the stimuli (TNF-alCHX, rotenone fand antimycin-A) had no effect, Heme-containing proteins in ‘mitochondria (cytochromes) and NOX enzymes in the cells are considered the major targets of CO due to the high affinity of CO for heme (Foresti and Motterlini, 2010). At the mitochondrial level, CO is known to inhibit complex IV (cytochrome oxidase), the terminal enzyme within the ETC, resulting in a significant transient burst of mitochondria-derived ROS (Of). Several reports support that a partial inhibition of complex IV may create a preconditioning state to protect cells against subsequent oxidative insults (Chin et al, 2007; Zuckerbraun et al, 20075 Kim et al, 2008). CORM-2 per se increased mitochondrial OF levels temporarily at 2 h of its incubation in MODE-K cells, whereas the total cellular ROS level measured at the same time point did not show any increase; the intracellular ROS lotecting probe carboxy-H2DCEDA can indoed be oxidized by ROS types, such as HaO2 and hydroxyl radical but not OF (Myhre et al, 2003). The early mitochondrial O}~ induetion by CORM-2 might contribute to its cytoprotective effect against (CORN under Oxia Ses Conctons TNF-a/CHX treatment, The induction of mitochondrial OF production by CORM.2 per se and the reduction by CORM.2 af mitochondrial O}- produced upon TNF-a/CHX, rotenone and antimycin-A treatment might be zelated to its lipid solubility. It seems plausible that the hydrophobic surface of CORM-2 enables its easy penetration through the outer and inner mitochondrial membrane, thus releasing CO inside the matrix. CORM.2 increased mitochondria-derived ROS as eatly as 30 min after its incubation with human airway smooth muscle eels (Taille etal, 2005). In MODE-K cells, measurable mitochondrial OF ‘was abserved only after 2h incubation, but this does not exclude the induction of mitochondrial Of by CORM-2 before 2 h. We have previously identified that signiicant ROS production by TNF-a in MODE-K cells can only be observed from 2b on, Jn contrast to other TECs, indicating that MODE-K cells are able to counteract the initial burst of oxidative steess up to this time point, ater which the antioxidant defense can no longer bbe maintained (Babu: et al, 2015b), The results of the current study therefore suggest a role of the lipid-soluble nature of CORM-2 in interfering with mitochondrial ROS signaling in ‘MODE-K IECS; further studies in other cell types are warranted to corroborate this phenomenon. Superoxide anion produced from NOX is converted into H,02 by superoxide dismutase (S0D)-1 (Cu/ZaSOD) in the eytoplasm, whereas Of” produced from mitochondria ae ether released as such into the cytoplasm by voltage-dependent anion channels (VDACs) or first converted into HO) by SOD-2 (MaSOD)/SOD-1 before diffusing across the mitochondrial membrane into the cytoplasm (Han et ala 2008), CORM-2 only partially reduced TNF-a/CHX-induced ritochondsial Of production, so that some mitochondrial OF” 1JH,Qr theoretically might leak into the cytoplasm in MODE'K cells, However, atleast 20; should then contribute tothe ROS, sigoal picked up with carboxy-HaDCEDA and this part should not be influenced by treatment with CORM-2, as originating from mitochondrial O}- not suppressed with CORM-2. As the increase in the DCF signal by TNE-a/CHX was fally abolished by CORM-2, this mitochondrial leakage does not seem to occur in MODE-K cells; this result further illustrates that CORM-2 abolishes NOX-derived ROS in MODE-K cell Mechanism of Action of CORM-401 during TNF-c/CHX-Induced Oxidative Stress imilar to CORM-A1, CORM-401 reduced TNF-aiCHX induced total cellular ROS production and cell death without any influence on TNF-a/CHX-induced mitochondrial OF production, Notably, CORM-401 per se did not have any effect ‘on mitochondrial OF production. CORM-AL attenuated the decrease in Wy and the increase in mitochondrial dysfunction by TNE-a/CHX, which might be related to its inhibitory effect ‘on mitochondrial respiration (Babu et al, 2015a). By contrast, CORM-401 did not influence these mitochondrial effects mediated by TNF-a, The inability of CORM-401 to modulate ‘TNF-a/CHX-induced mitochondrial O}- production and other ‘mitochondrial parameters seems to exclude mitochondria as 1 possible target influenced by CORM-401 during TNF-a Fetrury 2017 | our 8 | arteleabu et ‘TABLE 24 Qualtative summary of he effects of CORM-2 and CORM-0, ne antinysin-A treatment (CO-RMe under Oxane Ses Conctons sibated for 7h in basal conditions and ve. TNFu/CHX, rotenone ane Antinye Parameter Total ROS MiochondilOf~ Mitochondria MMochondial —Mitochondialf-Machondal OF etuncion membrane potenti Probes Carbony- —MIOSOX—_—MiloTacker Green! oo) MioSOXRed —MoSOX Red M.OCrDA fe itotacker Deop Red =; 7 . = = = oxidant pare é ‘ ‘ : ‘ EGRET beens = = - oxidant ‘= Not innuonced: ¥, Reduced: 4, Abolished, TABLE 25 | Queltatve summary a he effects of CORM-2, CORM-401 and CORM-AT vs. HzOp treatment 1 mM for 40min and 7.5 mM or 1. Parameter Probe Concentration of stimulos AM He0s Perio of ncubation 40min ‘= Not intuonced! ¥, Recuoad: #, From Bab ota 20 ‘CHX-induced cell death, Complexes 1 and IL of the ETC are the major mitochondrial ROS production sites during TNE /CHX-induced cell death in MODE-K cells (Babu et al. 2015). Consistent with the observation that CORM-401 does not influence TNF-a/CHX-induced mitochondrial OF” , CORM-401 was not able to reduce mitochondrial Of production by the complex I inhibitor rotenone, This result is probably related 10 the fact that rotenone-induced mitochondrial Of- is released into the mitochondrial matrix (Chen et al, 200%; Rodriguez Rocha et al, 2013), Indeed, as reported for CORM-A1 (Babu et al, 20158), CORM-401 was able to reduee antimycin-A- induced mitochondrial Of generation. This result could be attributed to the fact that the mitochondrial O}- generated by the Qi site inhibitor of complex ITT antimyein-A is reported 10 be fully (St-Pierre et al, 2002) or at least partially (Han et al, 2003b) released into the mitochondrial intermembrane space. Sill we have previously observed that mysothiazole, an inhibitor ‘of complex IIL at the Qo site, partially reduced ‘TNF-a/CHX. induced total ROS and cell death in MODE-K cells (Babu et al 2015b), implying that part of TNF-a/CHX-indueed ROS must also be released into the mitochondrial intermembrane space. ‘The lack of any effect of CORM-401 ys, TNF-a/CHX-induced ‘mitochondrial ROS might be related to near full use of CORM 401-derived CO in the cytoplasm to counteract NOX-derived Fron in Prarmacsiogy | wa tontersinog “ M02 Total ROS - Corbony-H,DCFOA Pre- + Co-ncubation + + ROS upon exposure to TNF-a. As NOX enzymesare not activated by antimycin-A, a higher amount of CO from CORM-401 might reach the mitochondrial intermembrane space upon exposure of MODE-K cells to antimycin-A, resulting in a more pronounced reduction of mitochondrial O$- than observed with CORM-2 treatment. As CORM-401 does not interfere with TNF-a/CHX. induced mitochondrial ROS, its inhibitory action on total cellular ROS induced by TNF-a/CHX treatment is exclusively related to inhibition of ROS derived from NOX, the second major source of TNF-a/CHX- induced ROS production. Inhibition of NOX by CO leading to decreased cytoplasmic O}- production has been previously reported (Bilban eta, 2006; Srisook et al. 2006; Wang etal, 20073 Kelsen et al, 2008). Differential Effects of CORM-2, CORM-A1 and CORM-401 during H202-Induced Cytotoxic Oxidative Stress ‘The main cellular ROS involved in redox signaling is probably 10, a8 O}~ produced by various intracellular sources ether spontaneously or through SODs dismutated into HO. H2O2 is the most abundant ROS in cells itis relatively stable and Jess toxic than other types of ROS and capable of difusing across membranes (Biener et al, 2006). Physiologically, low Fetrury 2017 | our 8 | arteleabu et endogenous levels of HO2 function as signaling molecules for the regulation of eukaryotic signal transduction but endogenous ‘overproduction of H,Q; is implicated in pathophysiological oxidative stress (Van de Bittner etal, 2010), Similarly, exogenous addition of higher concentrations of HO2 leads to oxidative stress and apoptotic cell death (Veal et al, 2007). The degree of effect of CORM-2 and CORM-401 under oxidative stress conditions in comparison with CORM-AL was therefore also investigated vs. H30,-induced ROS in MODE-K cells (see ‘Table 2B for a qualitative summary of the effects of CORM-2, CORM-401, and CORM-AI ys. H:0,). Incubation of MODE-K cells with CORM-2 or CORM-4O1 from 1 h before and during exposure to @ non-cytotoxic concentration of HO, (1. mM) {or 40 min similarly reduced Hy0,-induced intracellular total ROS levels, which is comparable to our earlier observation with, CORM-AI (Babu eta, 2015a). However, when tested vs. 7.5 mM. HO) for 1 h, inducing a similar level of cell death as TNF- aiCHX, but a much higher degree of total ROS, differences were “observed for CORM-401 vs, CORM-2 and CORM-AL. CORM. 401 isa water-soluble CO-RM with a half-life of CO release of 12: 1M min; itis able to release up to three CO per mole of compound, whereas CORM-2 and CORM-AL release one CO per mole of compound. Moreover, the release of CO is increased over time in the presence of biologically relevant oxidants, such as HO: (Payad-Kobeiss etal, 2016). This acceleration of CO release from CORM-401 when more oxidants are present probably explains ‘why CORM-401 is active in the presence of 7.5 mM H;03 when co-incubating the cells with the compound only during the 1 h exposure to HO3; the high degree of oxidative stress imposed by 5 mM H03 can be expected to maximally accelerate CO release from CORM-401. This effect does not accur with CORM-2 and. CORM-AI, which were ineffective with this treatment protocol Surprisingly, CORM-401 was also mildly effective when only pre- incubated for 1 h before exposure of the cells to 7.5 mM 110s. There is not a high ROS level in MODE-K cells before exposure to 7.5 mM 303; thus, CO release from CORM-401 is not accelerated. Stil, the amount of CO released from CORM-401 seems sufficient to provide some protection to the cells against 7.5 mM HO) after washout of CORM-401. We do not have a definitive explanation for this observation, which did not occur ‘with CORM-2 and CORM-A1. Both these compounds were only REFERENCES ‘An. 0 Ko,K.HOh,1-Y, Cho, Kin, W- 8 Lee K Jet (2001) Eieacy fas of enlonossopy in dextran ste sav induced weave eos in rats the evaluation ofthe efecto antioxidant by colonoscopy It. Colma Dis 6 74-18 doi 101007/003840000282 Anup, R, Aparna, V,Puliood A, and Balasubramanian, K. A (199). Surge stresand the sl nesting: ole of oxygen ie radials. Suen 125, 360-568, dot 1010160039 c06099)70209-6 Sabo, Lele, Gooner, V. Remsen. Nandenabel, Mottin al 2015) Andoxdast potential of CORMAL and resveratrol during TNFlphulcyteximide induced exidtve se and apoptosis in murine intestinal eptelal MODE-K cell Taxa APPL Pharmacel. 28, 161-178, dof 10 016 29.2015407 007 Babs, D, Lelreq G, Goosens, V., Vanden Berghe, Von Hamme, E Vandenabece, Py eta. (20150) Mitotondria and NADPH oxidases are the major sources of TNFalphafcyoheximide-induced oidatve ssn murine (CORN under Oxia Ses Conctons effective vs, 7.5 mM HO, upon 1 h pre-incubation plus 1h co-incubation. As no real iCORM401 can be prepared, some influence ofthe scaffold of CORM-401 cannot fully be excluded, CONCLUSION CORM.2 and CORM-401 show differential cytoprotective effects under oxidative stress conditions in MODE-K IECs, Both CORM-2 and CORM-401 show antioxidant and cytoprotective effects under oxidative stress associated with inflammation (TNE a/CHX). The cytoprotective effect of CORM-401 mitigates NOX- derived! ROS, whereas CORM-2 interferes with both NOX and ‘itochondria-derived ROS to protect MODE-K cells from TNE. a/CHX-induced cell death; the mitochondrial effect of CORM. 2 might be related to its lipophilicity. CORM-401 was more elfective than CORM-2 in protecting cells against oxidative stress and cell death, induced by a high concentration of exogenous HO}, This result might be related to the ability of CORM-401 to release more CO under oxidative conditions, suggesting that this compound may be effective under conditions of persistent oxidative stress such as in the case of acute and chronic GI disorders. Once probes are available allowing to analyze the subcellular location of released CO, it will be possible to investigate whether CO released from different CO-RMs can show a different subcellular trajectory. AUTHOR CONTRIBUTIONS DB and RL conceived and designed the experiments. DB performed the experiments. GL and RM contributed to the reagents, analytical tools and revision of the manuscript. DB and RL wrote the manuscript. All authors read and approved the final manuscript FUNDING ‘The study was supported by RL via grant BOFLO/GOA/024 from the Special Investigation Fund of Ghent University {nto epiteal MODE-K cel Co. Signal 27, 1141-1158, de 11016, clip 201502019 abs, Dy Metterins, R, and Lefbwre, R.A (2015). CO) and CO releasing raecles(CO-RMs)in acute gstointstnal inflammation, Br. .Pharmacok 172 1857-1573. doi 10.11 Ubph 12682 Baby, D, Soenen SJ, Raendonck K, Leclerc, De Backes, Mottin Ry ‘tal 201), TNP-alphacyloherimie-indacedoxkitive stress and apoptons {in marine testinal epiclial MODE K els, Curr: Phar Do 184814425, do 10247a/ise1212802181291 uregmian, N, Song, Bay, C. E, Papacomtantinoy, J, Esery 1M, ‘hd Chong. D-M. @005) "Tumor necrosis factoralphs and apoptosis gmabropulting hinne “1 control readive enygen apecs. Teme, rtochondil autophagy, and can N-terminal kinatelp 88 phosperylation ring ecrtiring enrol Orid. Mod Cell Lange 2, 297-306 ‘oi 1DAN6Loxim 259541 Bhattacharyya, Ay Chattopadhyay, R, Mita, Sy and Crows, 8. E (2018). ‘Onidatve stress: an ese factor in the pathogenesis of gastootesin! Fetrury 2017 | our 8 | arteleabu et rmucosil dieass, Physiol Rev, 9 329-354, doi, 10.152physrev00040, 20 Inert, G. P. Sehocsing, 1K. and Juha, T. (2006). Membrane wansport taf hydrogen peroaide.Boxhin Bioplys Ata 1758, 94-1008. do 10.1016, amen 20802015 Suan, ML ack, FH, Oterbein SL, ig, KA, Etre, ‘sl, 26) Carbon manos orchestrates protective reponse throwgh PAR. Immunity 24, 601-610 do 10,1016}immunt 200603 012 ‘iban Ms Hachem, , Weg, B, Chin, BY, Wagner, O, and Onerbsin (2008) Heme oxygenate and carbon monoxide inate homeostate signaling. Mol Met (Br 96, 267-279, doi: 101007500108. 07-0276-0 CCetinkaya, A, Babaloga,E, Kurutas, EB, Cali HL, Kantarceken, Band Buyukbese M.A, (205), Bene effects af N-acerycyteine on acte a Induced cols i ats Tool Esp. Mad. 206 131-139 do 101620/Dem, (Chen, Q, Vangucr FJ. Moghsddas,S, Hoppel, © and Leet, EJ. (2003) Production of reactive oxygen species by mitochondria: ental roiofcompex IL, Bok Chr 278, 36027-3403, do 101074 M4854200 Chin, BY. ang, G, Weg. B, Wang, H. Macdonald, T, Zhang. XC, eal (2007). Hyposi-ndacibe fico alpha stailzation by carbon monoxide esl in cytoprotective preconditioning. Proc Natl Acad. Sc. US.A. 104, 105-5114, do 101073} 0605611108 tk, .E, Naughton, P, Shure. S, Gree, CJ. Johnson, T-R, Mann B. Eta, (2003), Cardapeotzetiv ations a water sluble carbon monoid teleasing molec Cie Rex 98, <2-8 do 10.1610 RES ODDDSERSL 6567.08 Cotter Rosmele, C,Ronot, X, Levers, X, snd Mayo. . 2011) Crometre 'ssesaentf mitochondria wing resent praes. Ctometry 79, 05-405, oi 10.1002!202.21061 ‘Crook, SH, Mann, B. Ey Meijer A.J» Adams, H, Sav Seapens, Dy a (GOLD, [Ma(CO}s{S2CNMe(CH2CO2H)) anew waterseluble CO-tlesing ‘molecule lon Trans, 4, 420-4235, do 101039/cak10125k DeBacker, 0. lnc, E, Mancha, Leyaet L, Motels Rand Lefer CA. (2008), Water soluble CO relasing molecule reduce the development of pestopcrtv ie va molaton of MAPKINIO signaling ad reaction of Sahat tren Gi 58, 347-356 do 1113640 2008155381 de Winter I Yvan Nast, de Man, de Jonge, Brodenooed A. Seren. T, Cera (2005) Role of exitve stress the pathogenesis of epic eas in mice: Newasrortra. Mot 17, 251-261 ot: 10.1111/.1365.2982, 2004006183 Desmard M, Forest, R, Morin, D, Dagouasat M, Beats, A, Denamu Es ‘al (2012), Dilferentilanbacterial activity aguas Pseudomonas aeruginot by carhon monoxide-reeaing molec. Anti. Redox Sia. 153-163, le 10 108920112959, Fayad:Kbeiss, S. Ratoronsnteniny, J Dabie 1 Wikon J. Rogues, "A.M, Belcan, Ay tal (2016), Vascularandangogenic activi of CORM 401, an oxdant-senstve CO-rkeusing molecule. Bodhem, Phanmacol 102, (1-77 do: 10 1016p 2018.12014| Fore, Ry and Motta, R. (2010), Interaction of carbon monoside with ‘easton metal: evolutionary insights into deug target cscovery. Curt. Drag Taye 1, 1595-1604 do 1021741589401 11008011595, Guan, Y. Wovrll, RT Pets, T. A. and Moatrose, M. H. (200). ltestinal acho: reperason inj reverse a evel dame imaged vr Am J Phyl Gastron Liver Phyl 297, G187-C196- doe 1O1IS2/5p 503852008 an, Dy Antunes, Fy Canal R, Retork Dy and Cadenas, E2034). Voltage pendent anion channels coatol the reas ofthe superoxide anion fom Imiochondeia to cytosol. J Ba. Chet. 278, 5557-5563. da 1.1074 Mato269200 Hap, D., Canal, Reto, D, and Kapow, N. 2003). Efe of gation depletion on ses and topology of supcroide and hydrogen peroxide production in mitochondei, Mol Pharmacal 64 1135-1144 di 101124/a0, Horton, KL, Star, KM, Fonsea, SH Gv, and Kaley, 8 0. (208) ‘Mitochondria peneating peptides. Chm, Bik 15, 375-882. do: 10.0165, ‘heb! 200803015, ThX, DamersK, Zeng, Yo y Ores, and Wang, 2016) Toward carbon monoxide based therepcuti: cits dug lier and devlopabilty {es Phar, Si 105, 406-406 do 101016.p.2013 10.018 Fron in Prarmacsiogy | wa tontersinog (CO-RMe under Oxane Ses Conctons Tin S. Ray, R Ma and Johnson LR (2008), TNF alpbafyclohesimide induced ‘apoptosis in intestinal pitta cel reques Racl-eguate reactive oxygen Species Am. J. Phytol. Gastrointest. Liver Physiol. 298, G328-C357. da 1. 152ajpg.002192007| Kalen, S, Pate, BJ Pasker, LB, Vera, , Rimold, JM, Gadepal RS, tal (008). Hem oxygenase atenates angiotensin U-melitdmaperonise ‘rodction in cured mous thick ascending lop of Henle cell Am. Physio Rena Physiol 295, FLISA-F1L6, do 10.152aprenal0057.2008 Kim H., Loughran, B.A Rao J Billa, T- Ry and Zockerbrau, B.S (2008. ‘Carbon monoxide sates NF appa va ROS gnertion and AKt pathways to protet agaist cal death ofhepatocyes. A.J. Physiol. Gastro Liver Propo 295, G1A6-GIS2 do 10.1152 0105.20 ital M, Sidlgu, M.R. Tran, Redd, P and Mall A. B.(2014).Reatve ‘xyge spl in iat and ssw ary. Ania. Redox Steal. 20, 67 doi: 101089) 2012 519 i, Ry Clik, JE, Foret, , Sarathchanda, P, Mann, BE, and ‘Green, C.J, 02}. Carbon monoxide releasing molecles: characterization ‘of biochemical and vascular actives Cie. Res 90, EI7-E24. doi 10.1617 020.1050 Motel, Rand Foes, R (2014). Heme oxygenase asa tage for dro sconce As Reda Signal 20 1810-182, dl 10.1089/a5.2013 5658 Molin, Ry Mann, B. Ey and Forest, R. (2005). Therapetieappiations ‘of catbon monaxide-releasing molecules. Expert Opin Investig. Drags 14, 1305-1318 do 10A517/135407841411.1305 Mowclni, and Otterbein, LE. (200) The therapetic potential of carbon noride, Nat Re. Drag Discov 9728-748 do 1058/3278 Monel. R, Save, P Hammad, T> Bains S, Alberto, Ry Forest Ry ta (20056), CORM-AL’ a new pharmacologially active carbon monoxide releasing molecule. BASEB 19, 284-286. dor 101086, 04 269%¢ Mybue, ©. Andersen, JM, Aaenes Hand Fonnurn, (203) valuation of the probes 27 dichoroflvonacia dace, luminal and igen as indiestore of ective pees formation Biochem. Pharmacal 65, 15751542, dos 10.1016) Soing 2952(03)00085-2 Nishikawa, M, Oshitani, N Matsumoto, Nihigami 7 Artkawa, Ts and Toue, M. (2005. Aecumaltion of mitochondrial DNA mutation wih, loreal carcnogenei im eats colt. J. Caner 98, 331-357 do oss 02868 Oshitani, Ny Kitano, A Okabe, H Nakamora, S, Matsumoto, T and Kobayashi, K (1998). Location of superoxide anion gencation in human conic mucosa obtained hy bap. Gut, 936-938 do 10.1 1361G4t 347 9386 Pers, C, Bole, -P, Sergg | L, Dallas M.L, AL Owais, MM, Hettarachich, NT, etal. @015). Dire mechanisms underying the segulation of fon channels by carhon monosie-r J Pharmacol 172, 1546-1556 do 101110) bp. 12760 Ree, R, Nisenkor, Ay Mates, Z, and Bujnover, Y- (200), $-ASA and Iycopene decree the oridtive ses and inflaton inde yoni ts sith colts. Gastro 39, 514519, do 10 1007!0595.008 1336-7 Robinson, K. My Tones, M.S, Pehat My Monet, JS Ross M. Fy Hagen, TM, etal. (206) Seectvefluotesent maging of superoxide in vivo Using cthidlum based probes. Proc Nat. Acad Set USA. 108, 15088-1505. dae 10.073/pnan 01945105 Rodrigues Rocha, H, Garis Gand, A, ict, C, Li, S, Jones, [+ Chen, ‘tal (2013) Compartmentlized oxidative strewn dopamineeic cll death Induce by pesticides and comple Inhibitors: disne oes of superoxide anion and superoxide dismutases. Free Radic. Biok Med, 61, 370-388 do: 104016 ecradbiomed 201304001 Byte, SW, Alm, J, and Cho, A.-M. (206). Heme oxygenase-tcarbon ‘monaude rom bask scence to therapeutic applation. Pysol Rev. 86 583-650 do 10 1152/phystev 011.2005, Save, Forest, R, Mann, BE, Johnson, T-R, Green. and Motrin (2005). Carbon moncaide desing molecles (CO-RMS) attenuate the inflammatory reponse elsied by lipopolyeacharde in RAW2647 murine macrophages. BJ Pharmacol 14, 80010. dos 10103855. 70624 Svisook, K, Han. S, Choi HS 1, M. HL, Ueda Hs Kinn Ce a (2000) ‘CO fom enhanced HO activity or from CORM-2 inhibits both 0-20) and NO production and dawnegultes HO-t expression in LPS-stimulatd ‘macrophages Bloke, Pharmacol 71, 307-3, dls 101016) bep.2005.10.022 Fetrury 2017 | our 8 | arteleabu et St Pients[ Buckingham, JA, Robuck S.J, and Brand M.D. (202) Topology of supeonide production fom diferent sites inthe mitochondrial electron transport chan J Bl Chem 277 44784-44790, do 10.107)beM207217200 Sup, B, Sun, 2, in, and Chon, X (2008) CO releasing molecules (CORM.2) liberated CO attenuates leukocyte inflation inthe el ase of thermally injured mice J Bal Se 176-183. do 107150 98.176 Tl, Gx EF-Beana, [, Lanone. Sy Boczkowki Jo and Motrin, R. (2005) ‘Mitochondrial respiratory chain and NAD(PIH oxidase are targets for the mtiproliratve effect of carbon monoxide in human away smooth mos, Jol Che. 280, 25380-25340 do 101074} MS03512200 “Tahal T, Uchigama, Ky and Naito, Y. (2018) The therapeutic potedal of carbon ‘onoide fr inflammatory bows dicae, Digestion 9, 13-18 doi 101158) “ang. Chen. ang, H, and Nie D (2012) Short chain ty aes induced ‘uophgy serves an adaptive atgy for retarding mitochon mediated ‘popotie cel death. Cell Death ifr 8 603-618 do 10103 210.117 ‘an de Bites G. Cx Dubkewskaya, A Bertoni C: Ry and Changs. (2010) Tn vivo imoging of hydrogen peonide production in a murine tamer mode! witha chemescectve luminescent reporter. roc Not Acad Sc USA. 107, 21316-21821 do 10.1078/pas 1012868107 ‘ea E A, Day, A Mand Morgan, B.A. (207). Hyrogen prose sensing and igaling Mol Cll, 1-14, do 101016). melce 20070816 ‘idl, K, Grosjean, I, Evil J. P, Gespch, C. and Kaserian, D (1995) Immoctalzaton of mouse intestinal epibealclls by the SVAD-Lnge T gene Phenotypic and simunecharaerzaton a the MODE: Kell ine. lmao. ‘Method 166, 62-73 do 1010160022 175993)90329-6 Fronts in Phamacciogy | wn ontersinog ” (CORN under Oxia Ses Conctons Vii and Alves BL ME (2008). Pre-condioning Th Ly Queiogu. Gs induced ty carbon’ monexide provides neuronal protection against, apoptosis J. Newochem, 107, 375-384, dob 101011) 471-4159.2008 Wang. X, Wang ¥, Kim, H.P, Nakair, KByte. W. and Chol, ALM (2007) Carbon monoxide prec psi hyperona induced endothe poptsis by inhibiting reactive oxygen species formation. J Bil: Cher. 282, 1718-1725 do 101074} 84607610200 ‘Zhou, R, Yaa AS, Ment, P and Tchopp, |. (201). A role or mitochondria ‘in NURPS inflammasome astation, Natre 462, 221-225, do 1010587 atae8663 Zockerbraun, BS, Chin BY, Bilban, M Ava, JC, Rao Billa T. Re ta (2007) Carbon monexde signals ia ahibtion of cytochrome c oxidase and generation of mitochondrial active oxygen species FASEB [21 1099-108, oi 101096), 06-6644com Conflict of Interest Statement: The authors dsclire that the research was ‘conducted in the absence of any commercial or financial elaonships that could be construc as. potent conic of interest Copyright © 2017 Rabu, Leer, Mola and Lafebe. This i an opoacess “artic stibued wonder the terms of the Creative Commons Atrbution Liens (CC BY). The ws. dtrbutonar production ioe fons permited, provided the original author(s) or Mensor ae redid aed athe oil publication in his journal iced, acordance with accepted academic rate Nowe, dsribtion ‘vrcpodacton is pert whih des not comply with thw ferms Fetrury 2017 | our 8 | artele